Advancing Nematode Barcoding: a Primer Cocktail for the Cytochrome C Oxidase Subunit I Gene from Vertebrate Parasitic Nematodes

Molecular Ecology Resources (2013) doi: 10.1111/1755-0998.12082

Advancing nematode barcoding: A primer cocktail for the cytochrome c oxidase subunit I gene from vertebrate parasitic nematodes

SEAN. W. J. PROSSER,* MARIA. G. VELARDE-AGUILAR,† VIRGINIA. LEON-R EGAGNON† and PAUL.D.N.HEBERT* *Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario N1G 2W1, Canada, †Estacion de Biologıa Chamela, Instituto de Biologıa, Universidad Nacional Autonoma de Mexico, San Patricio, Jalisco 48980, Mexico

Abstract Although nematodes are one of the most diverse metazoan phyla, species identification through morphology is difficult. Several genetic markers have been used for their identification, but most do not provide species-level resolution in all groups, and those that do lack primer sets effective across the phylum, precluding high-throughput processing. This study describes a cocktail of three novel primer pairs that overcome this limitation by recovering cytochrome c oxidase I (COI) barcodes from diverse nematode lineages parasitic on vertebrates, including members of three orders and eight families. Its effectiveness across a broad range of nematodes enables high-throughput processing.

Keywords: barcoding, identiﬁcation, nematodes, primers Received 19 September 2012; revision received 9 November 2012; accepted 13 November 2012

high phenotypic plasticity (Coomans 2002; Nadler 2002), Introduction the absence of clear diagnostic characters (Wijova et al. Roundworms (Nematoda) are known to be among the 2005; Derycke et al. 2008) or their restriction to adults in most physiologically and ecologically diverse of meta- the numerous groups in which larvae are more often zoan phyla, occupying habitats from the deep sea to encountered (Anderson 2000). Given these constraints, deserts, and from the tropics to polar permafrost (Brown there is recognition that molecular techniques are critical et al. 1949, 1950; De Ley 2006; Dailey 2009; Asbakk et al. for taxonomic progress (Godfray 2002; Blaxter 2003). 2010; Vanreusel et al. 2010). The phylum includes free- Indeed, there are now online databases, such as NemA- living, parasitic, mutualistic, opportunistic and symbiotic TOL (http://nematol.unh.edu/), that are dedicated to taxa (Ott et al. 1991; Clarke 2008) and provides a useful organizing and storing ecological and molecular data of model system for the study of human diseases (Fire et al. nematodes. 1998; Barr 2005; Jadiya et al. 2011) and a tool for ecosys- Several genetic markers have been used for nematode tem surveillance (Sambongi et al. 1999; Marcogliese 2005; identification, including small and large subunit ribo- Ekschmitt & Korthals 2006; Wu et al. 2010; Denver et al. somal DNA (SSU and LSU respectively), the internal 2011; Hoess et al. 2011; Palm et al. 2011). However, nema- transcribed spacer (ITS) region of ribosomal DNA and todes are also a scourge as many species cause disease in cytochrome c oxidase subunit I (COI) (Blaxter et al. 1998; crops, livestock and humans (Hodda & Cook 2009; Man- Floyd et al. 2002; Subbotin et al. 2008; Elsasser et al. 2009; guin et al. 2010). Despite their importance, the taxonomy Ferri et al. 2009; Siddal et al. 2012). The ribosomal DNA of nematodes is poorly studied. Species-level identifica- small subunit (SSU) was the first marker used, and tion has traditionally relied on detailed morphological successfully delineated some nematodes but failed to analysis, a task requiring considerable expertise (Coo- completely explain previous observations based on mor- mans 2000) given the morphological conservatism and phology (Blaxter et al. 1998). As the use of SSU was small size of nematodes (Creer et al. 2010; Powers et al. expanded, it was discovered that the SSU barcode failed 2011). Aside from being time-consuming, morphology- to separate many species of nematodes and was better based identifications are often problematic because of suited for order or family-level discrimination (De Ley et al. 2005). The ribosomal DNA large subunit (LSU) Correspondence: Sean W. J. Prosser, Fax: 519-824-5703; was the second marker used in an attempt to develop E-mail: [email protected] a nematode phylogenetic classification system, but

© 2013 Blackwell Publishing Ltd Table 1 Nematode specimens used in this study. Taxa identified using morphology. Classification follows Hodda (2011) unless indicated by *, in those cases classification follows 2 Hodda (2007); ND: No Data PROSSER J. W. S.

Number of specimens studied (successfully Order Family Genus sequenced) Host species Locality Collection date

Panagrolaimida Rhabdiasidae Rhabdias sp. 1 1 (1) Smilisca Colima: Hwy Colima 7 July 2008

baudinii -Minatitlan AL. ET Panagrolaimida Rhabdiasidae Rhabdias sp. 2 4 (4) Rana sp. Nayarit: S of Hyw Barranca 25 June 2009 del Oro: Barranqueno~ bridge Panagrolaimida Rhabdiasidae Rhabdias sp. 3 5 (5) Rhinella marina Colima: Comala 7 July 2008 Panagrolaimida Rhabdiasidae Rhabdias lamothei 4 (4) Leptodeira sp. Colima: Hyw 98 Minatitlan 8 July 2008 -Manzanillo Rhabditida Molienidae* Oswaldocruzia sp. 9 (9) Phrynohyas Colima: Hyw Colima- 6 July 2008 venulosa Minatitlan 7 July 2008 Smilisca baudinii Colima: Hyw 98 Minatitlan -Manzanillo Rhabditida Diaphanocephalidae* Kalicephalus sp. 2 (2) Leptodeira sp. Colima: Comala 27 June 2009 Imantodes sp. Colima: Ixtlahuacan 24 June 2009 Spirurida Heterakidae Strongyluris sp. 1 (1) Trimorphodon Colima: Hwy 98 Minatitlan 8 July 2008 biscutatus -Manzanillo Spirurida Pharyngodonidae Ozolaimus sp. 5 (0) Ctenosaura sp. Colima: Hyw 54 Ixtlahuacan 8 July 2008 Spirurida Pharyngodonidae Parapharyngodon sp. 4 (4) Phrynohyas Colima: Hyw 98 Minatitlan 8 July 2008 venulosa -Manzanillo Spirurida Pharyngodonidae gen sp. 1 15 (10) Sceloporus sp. Jalisco: ND 25 July 2009 Spirurida Pharyngodonidae gen sp. 2 2 (2) Sceloporus formosus Veracruz: Hyw Xico 28 July 2004 Viejo- Matlalapa Spirurida Cosmocercidae Aplectana sp. 18 (17) Rana pustulosa Nayarit: S of Hyw Barranca 24 June 2009 Bufo sp. del Oro: Barranqueno~ bridge Leptodeira sp. Nayarit: Hyw Uzeta-La Gloria Colima: Comala Spirurida Onchocercidae Foleyellides sp. 11 (11) Rana pustulosa Nayarit: S of Hyw Barranca 24 June 2009 Rana psilonota del Oro: Barranqueno~ bridge 30 June 2010 © Jalisco: Zapopan: Barranca 03BakelPbihn Ltd Publishing Blackwell 2013 del rıo Santiago Spirurida Physalopteridae Physaloptera sp. 7 (7) Trimorphodon Michoacan: Hwy 200 5 July 2008 biscutatus between La placita and Maruata Spirurida Physalopteridae gen sp. 1 5 (5) Sceloporus sp. Jalisco: ND 25 June 2009 Spirurida Physalopteridae gen sp. 2 1 (1) Imantodes sp. Colima: Hyw Comala 25 June 2009 -Minatitlan Spirurida Physalopteridae Turgida sp. 1 (1) Didelphis virginiana Jalisco: Zapopan: Barranca 30 June 2010 del rıo Santiago PRIMERS FOR NEMATODE DNA BARCODING 3 requires the amplification of multiple regions to be effec- Materials and methods tive (De Ley et al. 2005; Subbotin et al. 2008). Similar studies using ITS revealed that a lack of phylum-wide Specimen collection primers combined with difficulties in aligning the extre- mely variable ITS sequences precluded its use as a Ninety-five adult nematodes collected in Mexico from universal nematode identification marker amenable to various reptilian, amphibian and mammalian hosts were high-throughput platforms (Floyd et al. 2002; De Ley analysed (Table 1). Each specimen was collected in et al. 2005). duplicate (i.e. from the same habitat within the same The mitochondrial gene cyctochrome c oxidase sub- host), with one stored in 95% ethanol for DNA extraction unit I (COI) has also been explored as a potential marker and the other cleared on a glass slide with undiluted on which to base a nematode phylogenetic classification glycerine to enable identification to family, genus or spe- system (Floyd et al. 2002; Elsasser et al. 2009). In addition cies level using morphological characteristics (Table 1). to being a mitochondrial gene, COI is translated into an evolutionarily conserved protein and thus has some Primer design advantages over SSU, LSU and ITS. However, COI is not immune to the inherent problems associated with nema- Cytochrome c oxidase subunit I (COI) sequences were tode barcoding. While the 5′ region of COI has been obtained from 56 mitochondrial genome sequences from shown to separate nematodes into proper species (Dery- nematodes in GenBank (Table 2) and aligned using cke et al. 2010), a phylum-wide primer set has yet to be online EBI CLUSTALW2 software (Larkin et al. 2007). A developed (De Ley et al. 2005). In this study, we report lepidopteran COI sequence was included in the align- the development of a primer cocktail which enables ment as a reference for locating the standard primer the recovery of COI barcodes from a broad range of binding sites (Folmer et al. 1994) for COI barcoding nematode parasites of vertebrates in a high-throughput (Hebert et al. 2003a,b). The forward and reverse primer manner and delivers species-level resolution. binding sites were excised from the 56 sequences and

Table 2 Nematode COI sequences used to design cocktail primers

GenBank Accession Species GenBank Accession Species

NC_008231 Agamermis sp. BH-2006 AJ556134 Necator americanus FJ483518 Ancylostoma caninum NC_003416 Necator americanus NC_003415 Ancylostoma duodenale GQ888716 Oesophagostomum dentatum GQ398121 Angiostrongylus cantonensis FM161883 Oesophagostomum quadrispinulatum GQ398122 Angiostrongylus costaricensis NC_001861 Onchocerca volvulus NC_007934 Anisakis simplex FN313571 Radopholus similis NC_001327 Ascaris suum NC_008640 Romanomermis culicivorax NC_004298 Brugia malayi NC_008693 Romanomermis iyengari FJ483517 Bunostomum phlebotomum EF175763 Romanomermis nielseni NC_009885 Caenorhabditis briggsae GU138699 Setaria digitata EU407789 Caenorhabditis briggsae NC_005941 Steinernema carpocapsae EU407793 Caenorhabditis briggsae DQ520860 Strelkovimermis spiculatus EU407804 Caenorhabditis elegans NC_008047 Strelkovimermis spiculatus NC_001328 Caenorhabditis elegans AJ558163 Strongyloides stercoralis EU407805 Caenorhabditis elegans GQ888717 Strongylus vulgaris EU407780 Caenorhabditis sp. GQ888718 Syngamus trachea GQ888721 Chabertia ovina GQ888720 Teladorsagia circumcincta HM773029 Chandlerella quiscali DQ520858 Thaumamermis cosgrovei NC_004806 Cooperia oncophora NC_008046 Thaumamermis cosgrovei GQ888712 Cylicocyclus insignis AM411108 Toxocara canis NC_005305 Diroﬁlaria immitis AM411622 Toxocara cati EU281143 Enterobius vermicularis AM412316 Toxocara malaysiensis NC_010383 Haemonchus contortus FJ664617 Toxocara vitulorum NC_008534 Heterorhabditis bacteriophora GU386314 Trichinella spiralis NC_008828 Hexamermis agrotis NC_002681 Trichinella spiralis GQ888722 Mecistocirrus digitatus GQ888719 Trichostrongylus axei GQ888714 Metastrongylus pudendotectus GQ888711 Trichostrongylus vitrinus GQ888715 Metastrongylus salmi NC_005928 Xiphinema americanum

Table 3 Primers used in this study. M13 tails are in lowercase bold

Primer Sequence (5′?3′) Reference

NemF1_t1 tgtaaaacgacggccagtCRACWGTWAATCAYAARAATATTGG This study NemF2_t1 tgtaaaacgacggccagtARAGATCTAATCATAAAGATATYGG This study NemF3_t1 tgtaaaacgacggccagtARAGTTCTAATCATAARGATATTGG This study NemR1_t1 caggaaacagctatgactAAACTTCWGGRTGACCAAAAAATCA This study NemR2_t1 caggaaacagctatgactAWACYTCWGGRTGMCCAAAAAAYCA This study NemR3_t1 caggaaacagctatgactAAACCTCWGGATGACCAAAAAATCA This study LCO1490_t1 tgtaaaacgacggccagtGGTCAACAAATCATAAAGATATTGG Folmer et al. 1994 HCO2198_t1 caggaaacagctatgacTAAACTTCAGGGTGACCAAAAAATCA Folmer et al. 1994 M13F TGTAAAACGACGGCCAGT Messing 1993 M13R CAGGAAACAGCTATGAC Messing 1993

phenograms for the two primer binding sites were gener- Table 4 PCR success rates of nematode cocktail primers ated using EBI CLUSTALW2. Both trees revealed three (C_NemF1_t1 + C_NemR1_t1) compared with Folmer primers + clusters (not shown) and the consensus sequence for (LCO1490_t1 HCO2198_t1) each cluster was used to design a primer cocktail consist- Number of PCR ing of one primer for each cluster (i.e. three forward and Primers positives Success Rate three reverse primers). The three primer sequences in each cocktail were tailed with modified M13 sequences C_NemF1_t1 + C_NemR1_t1 85/95 89.5% (Messing 1993) as described in Ivanova et al. (2007). LCO1490_t1 + HCO2198_t1 83/95 87.4% The three forward and three reverse primers were mixed in a 1:1:1 ratio to make the final forward + + Table 5 Sequencing success rates and trace quality scores of (C_NemF1_t1: NemF1_t1 NemF2_t1 NemF3_t1) and PCR products generated with nematode cocktail primers + + reverse (C_NemR1_t1: NemR1_t1 NemR2_t1 NemR3_t1) (C_NemF1_t1 + C_NemR1_t1) or Folmer primers cocktails (Table 3). (LCO1490_t1 + HCO2198_t1). Sequencing success rates were calculated by dividing the number of recovered sequences (after editing) by the total number of sequenced samples (i.e. 95) DNA extraction, PCR amplification and sequencing Average Success Rate Total DNA was extracted from whole nematodes using PHRED Success Rate (any sequence standard glass fibre methods (Ivanova et al. 2006). After Primers Score (661 bp only) over 100 bp) purification, 2 lL of DNA was added to a PCR reaction consisting of 6.25 lL of 10% D-(+)-trehalose dihydrate C_NemF1_t1 49 88.4% 88.4% (Fluka Analytical), 2.00 lL of Hyclone ultra-pure water + C_NemR1_t1 (Thermo Scientific), 1.25 lL of 10X PlatinumTaq buffer LCO1490_t1 44 65.2% 75.7% + l HCO2198_t1 (Invitrogen), 0.625 L of 50 mM MgCl2 (Invitrogen), 0.125 lL of each primer or primer cocktail, 0.0625 lLof 10 mM dNTP (KAPA Biosystems) and 0.060 lLof5U/lL 3730XL capillary sequencer (Applied Biosystems). Traces PlatinumTaq DNA Polymerase (Invitrogen) for a total were assembled and edited using CodonCode v. 3.0.1 reaction volume of 12.5 lL. Thermal cycling conditions (CodonCode Corporation, Dedham, Massachusetts). were 94 °C for 1 min, five cycles at 94 °C for 40 s, 45 °C Trace quality scores were calculated using KB Basecaller for 40 s, 72 °C for 1 min, followed by 35 cycles at 94 °C (ABI software) and trace statistics were calculated using for 40 s, 51 °C for 40 s, 72 °C for 1 min and a final exten- Sequence Scanner (Applied Biosystems). Sequences have sion at 72 °C for 5 min. The resulting amplicons were been deposited in BOLD (www.boldsystems.org) under â visualized on a 2% agarose E-gel 96 precast gel (Invitro- sample ID’s MXHEL359–MXHEL453 within the project gen) and bidirectionally sequenced using M13F and entitled: Parasitic nematodes from Mexican vertebrates M13R as sequencing primers (Table 3). (NEMNP) and in GenBank under accession numbers Cycle sequencing was performed using a modified KC130665 - KC130748. BigDye 3.1 Terminator (Applied Biosystems) protocol Since previous studies (Elsasser et al. 2009; Derycke (Hajibabaei et al. 2005). Cycle sequencing conditions et al. 2010) reported varying success in barcode recovery were 96 °C for 1 min followed by 35 cycles at 96 °C for with a commonly used primer set (LCO1490 and 10 s, 55 °C for 5 s, 60 °C for 2.5 min and a final extension HCO2198, Folmer et al. 1994), we compared the success of at 60 °C for 5 min. Sequencing was performed on an ABI sequence recovery with M13-tailed versions of LCO1490

© 2013 Blackwell Publishing Ltd PRIMERS FOR NEMATODE DNA BARCODING 5 and HCO2198 (Table 3) and our new cocktail. All PCR set, all 95 nematode samples were sequenced, even if an reagents were identical between the two primer sets, and amplicon was not visible on the E-gel. Sequences were the same DNA templates were employed. For each primer aligned using EBI CLUSTALW2, imported into MEGA5

Rhabdias sp. 2 KC130742 Rhabdias sp. 2 KC130740 Rhabdias sp. 2 KC130737 Rhabdias sp. 2 KC130748 Rhabdias sp. 1 KC130697 Rhabdias sp. 3 KC130739 Rhabdias sp. 3 KC130736 Rhabdiasidae Panagrolaimida Rhabdias sp. 3 KC130745 Rhabdias sp. 3 KC130741 Rhabdias sp. 3 KC130738 Rhabdias lamothei KC130744 Rhabdias lamothei KC130746 Rhabdias lamothei KC130747 Rhabdias lamothei KC130743 Kalicephalus sp. KC130691 Diaphanocephalidae Kalicephalus sp. KC130688 Oswaldocruzia sp. KC130711 Oswaldocruzia sp. KC130713 Oswaldocruzia sp. KC130715 Oswaldocruzia sp. KC130712 Molienidae Rhabditida Oswaldocruzia sp. KC130698 Oswaldocruzia sp. KC130687 Oswaldocruzia sp. KC130716 Oswaldocruzia sp. KC130704 Oswaldocruzia sp. KC130714 Strongyluris sp. KC130699 Heterakidae Aplectana sp. KC130733 Aplectana sp. KC130730 Aplectana sp. KC130720 Aplectana sp. KC130672 Aplectana sp. KC130674 Aplectana sp. KC130669 Aplectana sp. KC130668 Aplectana sp. KC130722 Aplectana sp. KC130665 Cosmocercidae Aplectana sp. KC130670 Aplectana sp. KC130729 Aplectana sp. KC130667 Aplectana sp. KC130666 Aplectana sp. KC130673 Aplectana sp. KC130671 Aplectana sp. KC130725 Aplectana sp. KC130723 Parapharyngodon sp. KC130695 Parapharyngodon sp. KC130705 Parapharyngodon sp. KC130700 Parapharyngodon sp. KC130689 Pharyngodonidae gen sp. 2 KC130735 Pharyngodonidae gen sp. 2 KC130734 Pharyngodonidae gen sp. 1 KC130724 Pharyngodonidae Pharyngodonidae gen sp. 1 KC130717 Pharyngodonidae gen sp. 1 KC130726 Pharyngodonidae gen sp. 1 KC130728 Pharyngodonidae gen sp. 1 KC130727 Spirurida Pharyngodonidae gen sp. 1 KC130731 Pharyngodonidae gen sp. 1 KC130719 Pharyngodonidae gen sp. 1 KC130732 Pharyngodonidae gen sp. 1 KC130718 Pharyngodonidae gen sp. 1 KC130721 Foleyellides sp. KC130679 Foleyellides sp. KC130686 Foleyellides sp. KC130683 Foleyellides sp. KC130678 Foleyellides sp. KC130675 Foleyellides sp. KC130681 Onchocercidae Foleyellides sp. KC130676 Foleyellides sp. KC130682 Foleyellides sp. KC130677 Foleyellides sp. KC130684 Foleyellides sp. KC130685 Physalopteridae gen sp. 1 KC130709 Physalopteridae gen sp. 1 KC130710 Physalopteridae gen sp. 1 KC130693 Physalopteridae gen sp. 1 KC130703 Physalopteridae gen sp. 1 KC130702 Physalopteridae gen sp. 2 KC130708 Turgida sp. KC130680 Physalopteridae Physaloptera sp. KC130701 Physaloptera sp. KC130694 Physaloptera sp. KC130707 Physaloptera sp. KC130696 Physaloptera sp. KC130690 Physaloptera sp. KC130692 Physaloptera sp. KC130706 0.07

Fig. 1 Neighbour-joining tree of COI barcode sequences generated by the nematode cocktail primers. A divergence of 2% or greater is indicative of a separate operational taxonomic unit. Codes following names of taxa refer to GenBank accession numbers.

(Tamura et al. 2011), and a neighbour-joining algorithm (e.g. Campagna et al. 2010; Clare et al. 2011; Kumar et al. (NJ) was used to generate a phenogram. 2012; Weigt et al. 2012). The barcode region of COI has delivered species-level resolution in certain nematode lineages (Derycke et al. 2010), but sequence recovery has Results proven difficult (De Ley et al. 2005). The primer cocktail PCR success rates (Table 4), as measured by the presence developed in this study appears to overcome this diffi- or absence of a visible amplicon on the E-gel, were very culty as it recovered full-length barcode sequences from similar with the Folmer primers (87%) and the new nematodes belonging to three orders and eight families primer cocktail (89%) (Fisher’s exact test, P = 0.8212). (Fig. 1), while 25% of the PCR products from Folmer However, there was a marked difference in sequence primers contained co-amplified sequences, perhaps quality and recovery (Table 5). The traces produced by reflecting poor binding with the target COI gene. More- the primer cocktail (n = 188) had a mean PHRED score over, the sequences recovered from our cocktail were of 49 (SD = 12), whereas those produced by the Folmer able to differentiate congeneric species, such as the four primers (n = 181) had a mean PHRED score of 44 species of Rhabdias, each from a different host and show- (SD = 12) (Student’s t-test, P = 0.0001). However, any ing consistent morphological differences as detected by traces with PHRED quality scores between 40 and 50 are Martınez-Salazar (2008) (Fig. 1; Table 1). Although we usually equally interpretable (personal observation). examined various taxa of nematodes parasitic of verte- More importantly, full-length barcodes (661 bp) were brates, further testing is required to validate the effective- recovered from 88% of the specimens with the new pri- ness of our primer set across the phylum. We examined mer cocktail, but from just 65% of reactions which representatives from three of the six currently recognized employed the Folmer primers (Fisher’s exact test. nematode orders parasitic on vertebrates (Hodda 2011), P = 0.0001). DNA barcodes were obtained from a total of all representatives of the Class Chromadorea. However, 84 specimens with 62 yielding full-length sequences with it is possible that our primer cocktail is effective across a both primer sets, while 12 were only recovered by the large diversity of nematodes because the primers were cocktail, and another 10 were fully recovered by the designed based on members of the Class Dorylaimea and cocktail but only partially (~500 bp) by the Folmer prim- other orders of Chromadorea of medical and veterinary ers. Every sequence generated by the cocktail allowed importance. An obvious next step will involve testing the assignment of its source specimen to an operational barcode recovery from representatives of other orders of taxonomic unit that agreed with its morphological iden- parasitic and free-living nematodes. tification (Martınez-Salazar 2008; Velarde-Aguilar, personal observation) (Fig. 1). Individuals from all genera Acknowledgements were successfully barcoded except Ozolaimus (Table 1); its failure may reflect poor DNA preservation since we This project was funded by the Government of Canada through observed that ethanol partially evaporated from the vial Genome Canada and the Ontario Genomics Institute by a grant that kept these specimens, and sequences were success- in support of the International Barcode of Life project (2008- OGI-ICI-03), by the CONACyT Red del Codigo de Barras de la fully recovered from members of closely related genera. Vida, Mexico (MEXBOL) in the form of a scholarship to MGVA, NSF grant DEB01613802 to Jonathan Campbell (University of Discussion Texas) and VLR and Proj. PAPIIT-UNAM No. IN-203911-3 to VLR. We thank Natalia Ivanova for helpful suggestions on pri- The Nematoda may be the most species-rich phylum of mer design and the manuscript, Angeles Romero-Mayen, animals, with approximately 27 000 described species Angelica Najar-Pacheco and Ma. Antonieta Arizmendi-Espinoza (Hugot et al. 2001; Hodda 2011), but taxonomic knowl- for their help in field collections. edge must progress significantly to validate this hypothe- sis. The ribosomal DNA small subunit (SSU) (Blaxter References et al. 1998), large subunit (LSU) (Subbotin et al. 2008) and internal transcribed spacer (ITS) region (Floyd et al. 2002; Anderson RC (2000) Nematode Parasites of Vertebrates: Their Development De Ley et al. 2005) have all been used as a tool for species and Transmission, 2nd edn. 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Vanreusel A, De Groote A, Gollner S, Bright M (2010) Ecology and biogeography of free-living nematodes associated with chemosyn- Data Accessibility thetic environments in the deep sea: a review. PLoS ONE, 5, e12449. DNA sequences: GenBank accessions KC130665 - KC130748 Weigt LA, Baldwin CC, Driskell A, Smith DG, Ormos A, Reyier EA and BOLD (www.boldsystems.org) sample ID’s MXHEL (2012) Using DNA barcoding to assess Caribbean reef fish biodiver- 359-MXHEL453. sity: expanding taxonomic and geographic coverage. PLoS ONE, 7, A spreadsheet with sampling and taxonomic details e41059. Wijova M, Moravec F, Horak A, Modry D, Lukes J (2005) Phylogenetic for each individual, GenBank accession numbers for its position of Dracunculus medinensis and some related nematodes DNA sequences, and its BOLD entry uploaded as online – inferred from 18S rRNA. Parasitology Research, 96, 133 135. supplementary material. Wu HC, Chen PC, Tsay TT (2010) Assessment of nematode community structure as a bioindicator in river monitoring. Environmental Pollution, DNA sequence alignment used to design primers, 158, 1741–1747. final DNA sequence alignment and phylogenetic data: BOLD (www.boldsystems.org) project ‘Parasitic nematodes from Mexican vertebrates’ (NEMNP). S.W.J.P. wrote the initial manuscript. M.G.V.A., V.L.R. and P.D.N.H. edited and contributed to the manuscript. M.G.V.A. and V.L.R. collected and identified all nematode Supporting Information specimens. S.W.J.P. designed nematode primers and Additional Supporting Information may be found in the online performed all molecular laboratory work and sequence version of this article: editing/interpretation. M.G.V.A. and V.L.R. analysed and Table S1 Specimen collection and taxonomic details. BOLD interpreted genetic-distance results. process ID’s and GenBank accession numbers are listed for each specimen.